Modifications of protein with carbohydrate or glycolipid are critical for human development. Congenital defects in asparagine-linked glycosylation, protein O-mannosylation, and glycosylphosphatidylinositol membrane anchoring of protein cause abnormal development of the eye, the nervous, and the musculoskeletal systems, as well as muscular dystrophy and seizures. The biochemical pathways for these protein modifications involve steps in which a sugar is transferred from a dolichol phosphate (Dol-P)-linked donor made on the cytoplasmic face of the endoplasmic reticulum (ER) membrane to a glycan or a polypeptide in the ER lumen. Seven mutations causing human glycosylation deficiencies occur in genes for Dol-P-sugar-utilizing glycosyltransferases (DPS-GT), unusual multi-spanning membrane proteins. The mechanisms of DPS-GT functions are unknown, so the effects of disease-causing mutations are obscure. The investigator hypothesizes that DPS-GT both translocates its Dol-P-sugar donor across the membrane and catalyzes lumenal glycosyltransfer, and are best treated as membrane transporters. Goals of the proposed research are to use strategies for expression of multispanning transporters in bacteria to purify and reconstitute representative DPS-GT, and test them for transport activity. In Specific Aim 1, the investigator will use two new bacterial systems for eukaryotic transporters to overexpress and purify DPS-GT. Proteins will first be expressed in the Gram positive bacterium Lactococcus lactis, which targets polytopic membrane proteins to its single membrane. DPS-GT is then expressed in Escherichia coli as fusions to green fluorescent protein, allowing a gel mobility shift assay that distinguishes folded from aggregated protein to be used to optimize conditions for expression of functional protein. DPS-GT will also be solubilized and purified using procedures proven effective for transporters, and their functionality will be verified by glycosyltransferase assay using synthetic acceptor glycans or peptides. In Specific Aim 2, the investigator will test the hypothesis that DPS-GT are bifunctional Dol-P-sugar translocating glycosyltransferases. Purified DPS-GT and Dol-P-sugar synthases will be reconstituted together in proteoliposomes containing Dol-P and preloaded with a soluble acceptor. Dol-P sugars will then be generated on the external face of the vesicle by adding sugar nucleotide substrate, and Dol-P-dependent glycosylation of lumenal acceptor upon supply of external substrate will be taken as evidence for Dol-P-sugar translocation by DPS-GT in the proteosomal membrane. Longer term goals are to determine whether translocation or glycosytransfer is affected in the DPS-GT mutated in congenital glycosylation diseases, and to explore how these defects can be corrected metabolically. PROJECT RELEVANCE: Attachment of carbohydrate-containing molecules to proteins is critical for human development, and congenital deficiencies in these additions result in severe abnormalities of the eye and nervous system, in muscular dystrophy, and in musculoskeletal defects. The biochemical pathways leading to these protein modifications include steps in which sugar molecules are moved across a cell membrane and then attached to protein, and enzymes involved in seven such steps are affected in congenital developmental diseases. Goals of this pilot study are to test a new hypothesis for how these enzymes work, and to determine how disease-causing mutations affect enzyme function.